This Article Full Text (PDF) References Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to Saved Citations Download to citation manager Request Permissions Request Reprints Add to My Marked Citations Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (49) Citing Articles via Google Scholar Citing Articles via Scopus Google Scholar Articles by Conway, B. R. Articles by Demaresti, K. T. Search for Related Content PubMed PubMed Citation Articles by Conway, B. R. Articles by Demaresti, K. T. Social Bookmarking                         What's this?

Quantification of G-Protein Coupled Receptor Internalization Using G-Protein Coupled Receptor-Green Fluorescent Protein Conjugates with the ArrayScan^TM High-Content Screening System

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Lisa K. Minor

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Jun Z. Xu

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Joseph W. Gunnet

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Robbin DeBiasio

Cellomics^TM Inc., Pittsburgh, Pennsylvania

Michael R. D'Andrea

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Richard Rubin

Cellomics^TM Inc., Pittsburgh, Pennsylvania

Richard DeBiasio

Cellomics^TM Inc., Pittsburgh, Pennsylvania

Ken Giuliano

Cellomics^TM Inc., Pittsburgh, Pennsylvania

Lubing Zhou

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Keith T. Demaresti

R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent homologous desensitization and internalization. Desensitization, defined as a decrease in the responsiveness to ligand, is accompanied by receptor aggregation on the cell surface and internalization via clathrin-coated pits to an intracellular endosomal compartment. In this study, we have taken advantage of the trafficking properties of GPCRs to develop a useful screening method for the identification of receptor mimetics. A series of studies were undertaken to evaluate the expression, functionality, and ligand-dependent trafficking of GPCR-green fluorescent protein (GFP) fusion conjugates stably transfected into HEK 293 cells. These GPCR-GFP expressing cells were then utilized in the validation of the ArrayScan^TM (Cellomics^TM, Pittsburgh, PA), a microtiter plate imaging system that permits cellular and subcellular quantitation of fluorescence in whole cells. These studies demonstrated our ability to measure the internalization of a parathy-roid hormone (PTH) receptor-GFP conjugate after ligand treatment by spatially resolving internalized receptors. Internalization was time- and dose-dependent and appeared to be selective for PTH. Similar results were obtained for a ß₂-adrenergic receptor (ß₂ AR)-GFP conjugate stably expressed in HEK 293 cells. The internalized GFP-labeled receptors were visualized as numerous punctate "spots" within the cell interior. An algorithm has been developed that identifies and collects information about these spots, allowing quantification of the internalization process. Variables such as the receptor-GFP expression level, plating density, cell number per field, number of fields scanned per well, spot size, and spot intensity were evaluated during the development of this assay. The method represents a valuable tool to screen for receptor mimetics and antagonists of receptor internalization in whole cells rapidly.

Journal of Biomolecular Screening, Vol. 4, No. 2, 75-86 (1999)
DOI: 10.1177/108705719900400207


CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				D. Haasen, S. Merk, P. Seither, D. Martyres, S. Hobbie, and R. Heilker Pharmacological Profiling of Chemokine Receptor-Directed Compounds Using High-Content Screening J Biomol Screen, January 1, 2008; 13(1): 40 - 53. [Abstract] [PDF]

				R. N. Ghosh, R. DeBiasio, C. C. Hudson, E. R. Ramer, C. L. Cowan, and R. H. Oakley Quantitative Cell-Based High-Content Screening for Vasopressin Receptor Agonists Using Transfluor(R)Technology J Biomol Screen, August 1, 2005; 10(5): 476 - 484. [Abstract] [PDF]

				B. K. Lundholt, V. Linde, F. Loechel, H.-C. Pedersen, S. Moller, M. Praestegaard, I. Mikkelsen, K. Scudder, S. P. Bjorn, M. Heide, et al. Identification of Akt Pathway Inhibitors Using Redistribution Screening on the FLIPR and the IN Cell 3000 Analyzer J Biomol Screen, February 1, 2005; 10(1): 20 - 29. [Abstract] [PDF]

				B. D. Schlag, Z. Lou, M. Fennell, and J. Dunlop Ligand Dependency of 5-Hydroxytryptamine 2C Receptor Internalization J. Pharmacol. Exp. Ther., September 1, 2004; 310(3): 865 - 870. [Abstract] [Full Text] [PDF]

				Z. Li, Y. Yan, E. A. Powers, X. Ying, K. Janjua, T. Garyantes, and B. Baron Identification of Gap Junction Blockers Using Automated Fluorescence Microscopy Imaging J Biomol Screen, October 1, 2003; 8(5): 489 - 499. [Abstract] [PDF]

				K. Moore and S. Rees Cell-Based Versus Isolated Target Screening: How Lucky Do You Feel? J Biomol Screen, April 1, 2001; 6(2): 69 - 74. [PDF]

				K. Berrada, C. L. Plesnicher, X. Luo, and M. Thibonnier Dynamic Interaction of Human Vasopressin/Oxytocin Receptor Subtypes with G Protein-coupled Receptor Kinases and Protein Kinase C after Agonist Stimulation J. Biol. Chem., August 25, 2000; 275(35): 27229 - 27237. [Abstract] [Full Text] [PDF]